Currently, more than about 60 kinds of viruses belonging to Flaviviridae (hereinafter abbreviated flaviviruses) are known, including Japanese encephalitis virus, West Nile virus, dengue 1-4 virus, yellow fever virus, St. Louis encephalitis virus, tick-borne encephalitis virus, Kunjin virus, Central European encephalitis virus, Kyasanur Forest virus, Murray Valley encephalitis virus, Omsk hemorrhagic fever virus, Powassan virus, Russian spring-summer encephalitis virus, Yokose virus, Apoi virus, and Aroa virus.
These flaviviruses have a genome of single-stranded (+) RNA and are similar to each other in terms of gene structure. The open reading frame (ORF) of the flavivirus genome encodes three structural proteins (capsid (C) protein, pre-membrane (prM) protein, which is the precursor for membrane (M) protein, and envelop (E) protein) and subsequent seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) from the 5′ terminus thereof.
These structural proteins and non-structural proteins of the flaviviruses are translated as single polyproteins; the polyproteins translated are then processed by the protease and NS3 protein having protease activity of host cells and the virus, resulting in the formation of a mature virion comprising the above-described three kinds of structural proteins.
It is known that many of the above-described flaviviruses infect to mammals including humans, and birds, via insects such as mosquitoes and ticks, and cause encephalitis and/or febrile symptoms. Originally, each of these flavivirus species is indigenous to a particular region; therefore, the endemic area of the infection had been limited. However, in recent years, due to development in traffics/distributions, climate change and the like, various flavivirus infections have expanded to places other than the original habitats of causal flaviviruses, posing an important problem with public health.
In preventing the expansion of viral infections, prevention with vaccines is effective. However, despite the fact that a large number of viruses belonging to Flaviviridae have been recognized as described above, only attenuated live vaccines for yellow fever virus and Japanese encephalitis virus infections and inactivated vaccines for Japanese encephalitis virus and tick-borne encephalitis virus infections are in practical application as vaccines for flavivirus infections. Particularly, attenuated live vaccines are useful as vaccines that are inexpensive and induce long-term immunity, but there are no approved live vaccines other than those described above.
In these circumstances, as a means of quickly developing novel attenuated live vaccines for various flavivirus infections, a strategy utilizing a chimeric flavivirus prepared by a gene engineering technique has recently been drawing attention.
For example, ChimeriVax™-JE is a chimeric flavivirus prepared by replacing the genes that encode two structural proteins (prM-E) of the yellow fever virus vaccine 17D strain with the corresponding genes of the Japanese encephalitis virus vaccine SA14-14-2 strain (see, for example, pamphlet for International Patent Publication No. 98/37911 and pamphlet for International Patent Publication No. 01/39802). ChimeriVax™-JE is attenuated to the extent that allows its use as a vaccine as a result of a plurality of amino acid mutations from the wild type that are present in the virus polyprotein corresponding to the E protein of the Japanese encephalitis virus vaccine SA14-14-2 strain (see, for example, Arroyo et al., J. Virol. 75:934-942, 2001).
Furthermore, on the basis of this technique for ChimeriVax™-JE, chimeric flaviviruses with dengue 1-4 viruses (ChimeriVax™-DEN (1-4)) (see, for example, pamphlet for International Patent Publication No. 98/37911 and pamphlet for International Patent Publication No. 01/39802) and a chimeric flavivirus with West Nile virus (ChimeriVax™-West Nile) (see, for example, pamphlet for International Patent Publication No. 2004/045529), which have a gene of the yellow fever virus vaccine 17D strain as the backbone, have also been developed.
These chimeric flaviviruses are also attenuated to the extent that allows their use as vaccines, with their attenuation resulting mainly from amino acid substitutions from the wild type that are present in the virus polyprotein corresponding to the prM-E protein.
However, it has been reported that the attenuation of ChimeriVax™-DEN1 results mainly from amino acid mutations in the E protein that occur during the passage of this chimeric flavivirus in cells for vaccine production (see, for example, Guirakhoo et al., J. Virol. 78:9998-10008, 2004). Furthermore, because re-infection with dengue viruses of different serotypes is likely as a cause of the onset of dengue hemorrhagic fever, this vaccine has not found a practical application.
Also, ChimeriVax™-West Nile has the attenuation promoted by artificially introducing an amino acid mutation in the E protein derived from the wild-type highly virulent West Nile virus NY-99 strain (see, for example, pamphlet for International Patent Publication No. 2004/045529).
As a chimeric flavivirus using a gene backbone other than yellow fever virus (YF-17D), a chimeric flavivirus prepared by replacing the gene that encodes the prM-E protein of dengue-4 virus with the corresponding gene of the West Nile virus NY99 strain (WN/DEN4 chimeric virus) has been reported (see, for example, Pletnev et al., Proc. Natl. Acad. Sci. USA 99:3036-3041, 2002).
Although the WN/DEN4 chimeric virus is attenuated compared to the parent strains thereof, i.e., West Nile virus and dengue-4 virus, the mechanism of the attenuation has not yet been fully elucidated, and the virus has not found a practical application as a vaccine.